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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 2020
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Glycerol tribenzoate
EC Number:
210-379-6
EC Name:
Glycerol tribenzoate
Cas Number:
614-33-5
Molecular formula:
C24H20O6
IUPAC Name:
1,3-bis(benzoyloxy)propan-2-yl benzoate
Test material form:
solid: flakes
Details on test material:
room temperature: off-white coloured flakes or granulate; > 80 °C: colourless to yellowish liquid
Batch no. 20118
CAS no. 614-33-5
Production date 28. Jul. 2020
Expiry date 15. Aug. 2022
Specific details on test material used for the study:
Batch no. 20118
Expiry date 15. Aug. 2022
Purity 98.1%

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 102
Species / strain / cell type:
S. typhimurium TA 1535
Species / strain / cell type:
S. typhimurium TA 1537
Metabolic activation:
with and without
Metabolic activation system:
S9
Note: each batch of S9 is characterized with a mutagen that requires metabolic activation by microsomal enzymes (e.g., benzo(a)pyrene).Note: each batch of S9 is characterized with a mutagen that requires metabolic activation by microsomal enzymes (e.g., benzo(a)pyrene).
Test concentrations with justification for top dose:
Experiment 1 Test item concentrations: 5000, 1500, 500, 150, 50 µg/plate
Experiment 1b Test item concentrations: 5000, 1500, 500, 150, 50 µg/plate
Experiment 2 Test item concentrations: 5000, 2500, 1250, 625, 313, 156, 78 µg/plate

On the day of the start of the first experiment, a stock solution containing 50 g/L of the test item in DMSO was prepared.
The stock solution was used to prepare the geometric series of the concentrations to be tested. The following nominal test item concentrations were prepared for experiment 1:
5000, 1500, 500, 150 and 50 µg/plate.

On the day of the start of the second experiment, a stock solution containing 50 g/L of the test item in DMSO was prepared.
The following nominal test item concentrations were prepared for experiment 2:
5000, 2500, 1250, 625, 313,156, 78 µg/plate.
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
other: 4-Nitro-1,2-phenylene diamine, 2-Amino-anthracene,
Details on test system and experimental conditions:
6.2.2 Origin and Culture
All Salmonella typhimurium strains were obtained from Trinova BioChem GmbH (batch: TA98: 5465D, TA100: 5439D, TA102: 5420D, TA1535: 5484D, TA1537: 5450D) and were stored as lyophilizates in the refrigerator at 2 – 8 °C.

On the day before the start of each experiment, a nutrient broth (Oxoid nutrient broth no. 2) was inoculated with one lyophilizate per strain at 3:30 pm (exp. 1) and 3:20 pm (exp. 1b and 2).
These overnight cultures were placed in the heating chamber at 37 ± 1 °C for 16 hours and 30 min. (exp. 1) and 16 hours and 40 min. (exp. 1b and 2).
For the last two hours the overnight cultures were shaken on an orbital shaker (150 rpm) at 37 ± 1 °C.
Afterwards, the overnight cultures were ready for use in the experiment.
During the test, the overnight cultures were stored at room temperature (20 ± 5 °C) to prevent changes in the titre.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

For TA102, the experiment 1 was invalid, since the spontaneous revertants were higher than historical control data and the f(I) values of the positive controls were not > 2 in the presence and absence of metabolic activation.

The experiment was repeated for TA102 as experiment 1b and was valid.

The test item showed no precipitates andno signs of toxicity towards the bacteria strainson the plates at any of the concentrationsin boththe presence and the absence of metabolic activation in all evaluated experiments.

The results of all experiments showed that none of the tested concentrations showed a significant increase or dose-related increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

 

Based on the results of this study it is concluded thatGlycerol tribenzoate (GTB)is not mutagenic in theSalmonella typhimuriumstrains TA98, TA100, TA102, TA1535 and TA1537 in the presence and absence of metabolic activation under the experimental conditions in this study.

Applicant's summary and conclusion

Executive summary:

Based on the results of this study it is concluded thatGlycerol tribenzoate (GTB)is not mutagenic in theSalmonella typhimuriumstrains TA98, TA100, TA102, TA1535 and TA1537 in the presence and absence of metabolic activation under the experimental conditions in this study.